The objective of the proposed studies is to examine the regulation of the death of CD4 T cells due to lack of growth or survival factors (termed passive cell death - PCD) and the death of such cells after or during vigorous activation and proliferation (termed activation-induced cell death - AICD); and the roles of these pathways of apoptosis in regulating specific immune responses and in maintaining homeostasis in the immune system. The hypothesis underlying the project is that antigen, growth factors and costimulators have different effects on death pathways at different stages of lymphocyte activation, and this is a major determinant of the functional consequences of these signals and, therefore, the resultant immune response. There are two specific Aims. Aim1) to elucidate the physiological roles and regulation of pathways of apoptosis. Using an experimental system of transferring T cells expressing a transgenic antigen receptor into normal recipients and exposing these cells to antigen in vivo, the principal investigator will determine when after antigen exposure the specific T cell expresses proteins that regulate PCD or AICD, and how this is influenced by the nature of immunization and the frequency of responding cells, and how it correlates with cell cycling, apoptosis and differentiation. The roles of costimulators and cytokines, notably IL-2 will be examined using specific antagonists and CD28 and IL-2 knockout mice. The roles of passive cell death and AICD in regulating the immune response will be examined using TCR transgenic T cells that constitutively express Bcl-2/Bcl-x or are defective in Fas/FasL. Parallel experiments will be done in vitro, under conditions where the T cell stimuli can be controlled precisely and accurate correlations with apoptosis established. Aim 2) to investigate the biochemical regulation of AICD. The sensitivity of T cells to AICD is regulated by T cell activation, cycling and IL-2. The biochemical mechanisms of this regulation will be defined, with emphasis on: 1) expression of FasL and the components of the Fas signaling complex; 2) assembly of the signaling complex; and 3) transcription and expression of the anti-apoptotic proteins, particularly the inhibitors FLIP, Bcl-2/Bcl-xL, and the Fas associated phosphatase.